16S rRNA gene amplification and sequencing.

Genomic DNA for each isolated bacterial strain was extracted by using a GenElute bacterial DNA kit (Sigma-Aldrich), following the Gram-positive protocol according to manufacturer’s instructions, and used as a template for PCR. The 16S rRNA gene in the bacterial strains was amplified using modified versions of the universal 27FYM and 1492R 16S rRNA gene primers with built-in redundancies (see Text S2), using Phusion Green Hot Start II High Fidelity PCR Master Mix (Thermo Fisher Scientific). PCR conditions were as follows: initial denaturation at 98°C for 30 s; followed by 25 cycles of 98°C for 10 s, 47°C for 45 s, and 72°C for 30 s; followed in turn by a final extension at 72°C for 10 min. The amplified products were purified using Macherey-Nagel NucleoSpin gel and PCR Clean-Up (Fisher Scientific), followed by Sanger sequencing using the universal M13 primers M13 uni(-21) and M13 rev(-29) (Eurofins Genomics). For certain samples, internal primers for the 16S rRNA gene (342R, 357F, 519R, 907R, 926F, 1100R, 1114F, and 1392R) were also used for sequencing to improve the sequence coverage accuracy. All primer sequences have been provided in Text S2.

Modified version of the universal primers of the 16S rRNA gene. Download Text S2, DOCX file, 0.01 MB.